Project Summary: Accurate Molecular Decision Making during Protein Biogenesis Accurate protein biogenesis is essential for the generation and maintenance of a functional proteome. Our long term goal is to understand the molecular mechanisms by which diverse protein biogenesis pathways in the cell accurately select nascent protein substrates and ensure their correct folding, localization, and maturation. Two major components define our research program in this grant cycle. First, we will use a combination of biochemical, biophysical, and in vivo experiments to decipher the mechanisms by which nascent proteins emerging from the ribosome are selected and processed by ribosome-associated protein biogenesis factors (RPBs) in the crowded space of the ribosome tunnel exit. These studies will include a new co-translational membrane protein targeting pathway mediated by SecA, enzymes mediating N-terminal methionine excision on nascent proteins in bacteria, and co-translational protein targeting mediated by SRP in the mammalian system. In addition to studying the biochemical and biophysical mechanisms of the individual protein biogenesis pathways, we will also elucidate how each of these factors coordinates with other RPBs in space and time during ongoing translation, and how this coordination reshapes the efficiency and fidelity of the individual pathways. Second, we will decipher the mechanisms by which aggregation-prone membrane proteins are effectively protected and facilely guided to the target membrane during their post-translational targeting. These studies will use two membrane protein biogenesis pathways as models: (i) an ATP-independent chaperone cpSRP43, which allows us to decipher, at biophysical resolution, the molecular mechanisms by which a small chaperone effectively protects multi-pass membrane protein clients and achieves spatiotemporal regulation of its client interactions in the absence of ATPase cycles or cochaperones; (ii) the guided-entry of tail-anchored proteins (GET) pathway, which provides an excellent system to decipher how a multi-component Hsp70- cochaperone cascade protects, funnels, and triages nascent membrane proteins during their targeted delivery. Investigation of the GET pathway will also allow us to gain insights into the design and organizational principles of analogous chaperone networks in the cell. The proposed experiments will not only generate high resolution understandings of the individual protein biogenesis pathways, but also establish valuable tools, reagents to explore the action of other protein biogenesis machineries. Most importantly, this research will generate important conceptual frameworks to understand how nascent proteins are accurately selected into their appropriate biogenesis pathways in the crowded cytosolic environment.